In recent years, it has become a common practice to detect nucleic acid sequences by hybridization as an efficient means of diagnosis of genetic diseases, cancer, infection, and other diseases.
The target base sequence is often only a very small portion of the detection subject nucleic acid; and when an oligonucleotide probe with an end labeled with a non-radioactive marker or an oligonucleotide probe labeled with a radioisotope is used, nucleic acid sequence detection is sometimes difficult due to insufficient sensitivity and other problems. Much effort has, therefore, been made to improve probe detection system sensitivity (W087/03622).
A published means of sensitivity improvement includes amplifying the target nucleic acid sequence to be detected with the use of DNA polymerase (Japanese Patent Unexamined Publication No. 274697/1986, hereinafter sometimes abbreviated as PCR). This method, however, requires frequent heating and cooling for repeated cycles of primer annealing, elongation and denaturation, which in turn demands specially designed equipment or much labor. Also, this method uses two oligonucleotides as primers. Amplification by DNA polymerase may occur even when these oligonucleotides have annealed nonspecifically, and stringent primer specificity is required.
Another published means is the amplification method using DNA ligase (W089/12696, Japanese Patent Unexamined Publication No. 2934/1990). This method, however, involves nonspecific amplification by DNA ligase blunt-end ligation. To avoid this phenomenon, the method using three or more sets of oligonucleotide probes has been published (W089/12696), but this method requires high cost because a large number of probes are necessary. Another published method uses a combination of DNA ligase and DNA polymerase (W090/01069). However, this method also poses a problem of high cost because it requires at least four oligonucleotides in two sets. Another method requires three oligonucleotides, two of which require several dozens of nucleotides, resulting again in high cost.
Also, it is a well-known fact that RNA is produced from DNA by the action of RNA polymerase, and a nucleic acid amplification method using RNA polymerase has been published (W089/01050). In this method, however, satisfactory amplification cannot be achieved solely by transcription and amplification with RNA polymerase. Therefore, the resulting RNA again undergoes the action of reverse transcriptase to produce DNA. Generally, transcription to DNA by reverse transcriptase is faulty, since it often accompanies reading errors in comparison with DNA replication by DNA polymerase. There is a known method in which only the probe which has correctly hybridized to the target nucleic acid is amplified [BIO/TECHNOLOGY vol. 6, 1197 (1988)]. This method, however, has a shortcoming in that the blank value increases because the probe bound by nonspecific reaction is also amplified.